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Abstract:

A fuel cell has an anode and a cathode with anode enzyme disposed on the
anode and cathode enzyme is disposed on the cathode. The anode is
configured and arranged to electrooxidize an anode reductant in the
presence of the anode enzyme. Likewise, the cathode is configured and
arranged to electroreduce a cathode oxidant in the presence of the
cathode enzyme. In addition, anode redox hydrogel may be disposed on the
anode to transduce a current between the anode and the anode enzyme and
cathode redox hydrogel may be disposed on the cathode to transduce a
current between the cathode and the cathode enzyme.

Claims:

1-6. (canceled)

7. An electrochemical cell for positioning in a biological system
comprising oxygen, comprising: a substrate comprising a first face and a
second face; an anode disposed on the first face of the substrate,
wherein the anode comprises an anode enzyme and a mediator, wherein the
enzyme and the mediator are crosslinked; and a cathode disposed on the
second face of the substrate.

8. The electrochemical cell according to claim 7, wherein the mediator is
a metallocene derivative.

9. The electrochemical cell according to claim 8, wherein the metallocene
derivative is ferrocene.

10. The electrochemical cell according to claim 7, wherein the mediator
comprises a transition metal complex.

12. The electrochemical cell according to claim 7, wherein the anode
comprises a conductive material selected from the group consisting of
gold, carbon, platinum, ruthenium dioxide and palladium.

13. The electrochemical cell according to claim 12, wherein the anode
comprises carbon or gold.

14. The electrochemical cell according to claim 7, wherein the substrate
surface is roughened.

15. The electrochemical sensor according to claim 7, wherein the anode
enzyme is a glucose-responsive enzyme.

16. The electrochemical cell according to claim 15, wherein the
glucose-responsive enzyme is glucose oxidase or glucose dehydrogenase.

17. The electrochemical cell according to claim 15, wherein the
glucose-responsive enzyme further comprises an enzyme cofactor.

18. The electrochemical cell according to claim 17, wherein the enzyme
cofactor is pyrroloquinoline quinone (PQQ).

19. The electrochemical cell according to claim 7, wherein the cathode
comprises platinum.

20. The electrochemical cell according to claim 7, wherein the cathode
further comprises a cathode enzyme.

21. The electrochemical cell according to claim 20, wherein the cathode
enzyme is an enzyme selected from the group consisting of tyrosinase,
horseradish peroxidase, soybean peroxidase, laccase and cytochrome C
peroxidase.

22. The electrochemical cell according to claim 21, wherein the cathode
enzyme is laccase or cytochrome C peroxidase.

23. The electrochemical cell according to claim 7, wherein at least a
portion of the electrochemical cell is configured to be subcutaneously
positioned in a subject.

24. The electrochemical cell according to claim 7, wherein the
electrochemical cell further comprises a mass transport limiting membrane
disposed over the anode and cathode.

25. The electrochemical cell according to claim 24, wherein the mass
transport limiting membrane is configured to limit contact of
macromolecules of 5000 daltons or greater with the anode and cathode.

26. The electrochemical cell according to claim 24, wherein the mass
transport limiting membrane is a hydrogel.

27. The electrochemical cell according to claim 26, wherein the hydrogel
comprises at least 20 wt % fluid when in equilibrium with
analyte-containing fluid.

Description:

[0001] This application is a continuation of application Ser. No.
11/277,696, now U.S. Pat. No. 7,238,442, which is a divisional of
application Ser. No. 10/385,069, filed on Mar. 10, 2003, now U.S. Pat.
No. 7,018,735, which is a continuation of application Ser. No.
09/961,621, filed Sep. 24, 2001, now U.S. Pat. No. 6,531,239, which is a
continuation of application Ser. No. 09/203,227, now U.S. Pat. No.
6,294,281, which claims priority to provisional application No.
60/089,900.

FIELD OF THE INVENTION

[0002] The present invention is, in general, directed to fuel cells and
methods of their manufacture and use. More particularly, the present
invention relates to fuel cells capable of operation by electrolyzing
compounds in a biological system and methods of their manufacture and
use.

BACKGROUND OF THE INVENTION

[0003] There is interest in a variety of techniques for providing
intermittent or continuous electrical power from a power source that
utilizes constituents of the environment. In the context of devices
implanted in a human or animal, there is a desire to find an energy
source that utilizes the body's own chemicals for providing electrical
power to the device. This typically includes a mechanism for converting
energy stored in chemical compounds in the body to electrical energy.
Such devices have been difficult to prepare and implement.

[0004] In outdoor situations, solar energy, wind energy, and mechanical
vibrations have been used to provide power from the environment. However,
because of the diffuse nature of these sources of energy, devices with
relatively large footprints are needed to provide the desired energy.
Furthermore, these sources of energy are often intermittent and may not
be available in all situations. Another potential source of energy is
available from chemical energy stored in plants or their residue.

[0005] Electrochemical fuel cells have been developed to convert energy
stored in chemical compounds to electrical energy. After nearly 50 years
of research and development, however, only the hydrogen anode/oxygen
cathode fuel cell operates at ambient temperatures. Fuel cells that
operate using organic compounds have not been developed, at least in
part, because the surfaces of electrocatalysts for the oxidation of
organic compounds have not been stabilized. Fouling by intermediate
oxidation products, that are strongly bound to the active sites of the
catalysts, causes loss of electrocatalyst activity. Thus, there is a need
for the development of electrochemical fuel cells that have
electrocatalysts that are resistant to fouling and that can operate using
compounds found in biological systems.

SUMMARY OF THE INVENTION

[0006] Generally, the present invention relates to fuel cells that operate
using fuels from biological systems. One embodiment is a fuel cell having
an anode and a cathode.

[0007] Anode enzyme is disposed on the anode and the anode is configured
and arranged to electrooxidize an anode reductant in the presence of the
anode enzyme. Likewise, cathode enzyme is disposed on the cathode and the
cathode is configured and arranged to electroreduce a cathode oxidant in
the presence of the cathode enzyme. In addition, anode redox hydrogel may
be disposed on the anode to transduce a current between the anode and the
anode enzyme and cathode redox hydrogel may be disposed on the cathode to
transduce a current between the cathode and the cathode enzyme.

[0008] Electrical energy is produced in the fuel cells of the present
invention as a biological fluid containing the anode reductant, such as,
for example, sugars, alcohols, carboxylic acids, carbohydrates, starches,
and cellulose, and the cathode oxidant, such as, for example, O2,
flows through the cell. The electrical energy produced by the fuel cell
can be stored or used to power an attached device.

[0009] Another embodiment of the invention is a method of generating
electrical power in a biological system by inserting an anode and a
cathode into the biological system. A biochemical anode reductant is
electrooxidized on the anode in the presence of an anode enzyme. And a
cathode oxidant is electrodreduced on the cathode, spaced apart from the
anode, in the presence of a cathode enzyme.

[0010] The above summary of the present invention is not intended to
describe each disclosed embodiment or every implementation of the present
invention. The Figures and the detailed description which follow more
particularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention may be more completely understood in consideration of
the following detailed description of various embodiments of the
invention in connection with the accompanying drawings, in which:

[0012] FIG. 1 is a not-to-scale cross-sectional view of one embodiment of
a fuel cell, according to the invention;

[0013] FIG. 2 is a perspective view of a second embodiment of a fuel cell,
according to the invention;

[0014] FIG. 3 is a perspective view of a third embodiment of a fuel cell,
according to the invention;

[0015] FIG. 4 is a perspective view of a fourth embodiment of a fuel cell,
according to the invention; and

[0016]FIG. 5 is a perspective view of a fifth embodiment of a fuel cell,
according to the invention.

[0017] While the invention is amenable to various modifications and
alternative forms, specifics thereof have been shown by way of example in
the drawings and will be described in detail. It should be understood,
however, that the intention is not to limit the invention to the
particular embodiments described. On the contrary, the intention is to
cover all modifications, equivalents, and alternatives falling within the
spirit and scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0018] The present invention is believed to be applicable to fuel cells
and methods of manufacture and use. In particular, the present invention
is directed to fuels cells capable of using compounds from biological
systems as fuel and methods of manufacture and use. For example, fuel
cells can be made that oxidize biochemicals available in the body of an
animal, in a plant, or in plant residue. Examples of oxidizable
biochemicals include sugars, alcohols, carboxylic acids, carbohydrates,
starches, and cellulose. The fuel cell can be implanted in a portion of
the animal or plant where a fluid, such as blood or sap, flows or the
fuel cell can operate utilizing tissue or fibers, particularly,
cellulose, as a fuel. While the present invention is not so limited, an
appreciation of various aspects of the invention will be gained through a
discussion of the examples provided below.

Fuel Cell

[0019] The fuel cells of the invention typically operate using, as fuel,
compounds available in a biological system. Fuel cells of the invention
can be operated in a variety of biological systems. For example, a fuel
cell may be configured for implantation into a person or animal to
operate an electrical device, such as a pacemaker, a nerve growth
stimulator, a nerve stimulator for relief of chronic pain, a stimulator
for regrowth of bone or other tissue, a drug-release valve or microvalve,
or a fluid-flow control valve, such as a valve in a duct or in the
urinary tract. Another example of a fuel cell for use with a biological
system is a fuel cell that provides electricity from a plant, tree, plant
residue, or the like. Typically, the fuel cells operate as a biological
fluid, such as, for example, blood or sap, flows through the fuel cell.
This provides a replenishing source of reactants for the fuel cell.

[0020] The fuel for the operation of the fuel cell may be provided by
compounds in blood, sap, and other biological fluids or solids. Such
compounds may include, for example, sugars, alcohols, carboxylic acids,
carbohydrates, starches, cellulose, and dissolved or complexed oxygen
(e.g., oxygen complexed with a biomolecule such as hemoglobin or
myoglobin). Examples of compounds for electroreduction or
electrooxidation in the operation of a fuel cell in an animal include
glucose or lactate at the anode and oxygen, dissolved as molecular oxygen
or bound in hemoglobin or myoglobin, at the cathode.

[0021] A fuel cell 100 of the invention for use in a biological system
includes an anode 102 and a cathode 104, as illustrated in FIG. 1. The
anode 102 and cathode 104 are separated to avoid shorting. Optionally,
separation between the anode 102 and cathode 104 is accomplished using
one or more spacers 103. The spacers 103 are often permeable, porous,
microporous, and/or fibrous. Alternatively, the spacers 103 may have gaps
to allow fluid to flow through the spacers 103. In some embodiments, the
spacers 103 may be ion selective membranes. Suitable materials for the
spacers 103 include, for example, polyamides (e.g., nylon), polyesters
(e.g., Dacron®), a cation exchange membrane (e.g., Nafion®), an
anion exchange membrane, porous polyolefins, polyimides, polyethers, and
polyurethanes.

[0022] An anode electrolysis layer 106 is formed on at least a portion of
the anode 102.

[0023] The anode electrolysis layer 106 typically includes an anode redox
polymer and an anode enzyme. Likewise, the cathode 104 has a cathode
electrolysis layer 108, typically including a cathode redox polymer and a
cathode enzyme, formed on at least a portion of the cathode 104. More
than one redox polymer and/or more than one enzyme can be used in each
electrolysis layer. In some embodiments, the anode electrolysis layer 106
and/or cathode electrolysis layer 108 are covered by a non-fouling
coating 109.

[0024] The enzyme in each electrolysis layer typically catalyzes an
electrochemical reaction of a cathode oxidant or anode reductant.
Usually, the anode reductant is electrooxidized at the anode 102 and the
cathode oxidant is electroreduced at the cathode 104. The redox polymer
transduces a current between the cathode oxidant or anode reductant and
the respective electrode. In general, the cathode oxidant and anode
reductant are provided within the biological system. In one embodiment,
the cathode oxidant is oxygen and the anode reductant includes sugars,
alcohols, and/or carboxylic acids. The fuel cell optionally includes
enzymes that break down more complex molecules, such as, for example,
starches and cellulose, into simpler components, such as sugars,
alcohols, and/or carboxylic acids.

[0025] The physical dimensions, as well as the operational parameters,
such as the output power and voltage, are, at least in part, a function
of the components of the fuel cell. The open circuit voltage of the fuel
cell can range from, for example, 0.5 volts to 1.2 volts, however, the
fuel cells of the invention can also produce larger or smaller voltages.
The voltage at the maximum power point can range from, for example, 0.4
to 0.8 volts. In addition, two or more fuel cells may be combined in
series and/or in parallel to form a composite fuel cell with a larger
voltage and/or current. The volumetric output power density of the fuel
cell can range from, for example, about 0.5 mW/cm3 to about 5
W/cm3, however, fuel cells can also be formed with higher or lower
volumetric output power density. The gravimetric output power density can
range from, for example, about 5 mW/g to about 50 W/g, however, fuel
cells can also be formed with higher or lower gravimetric output power
density. The output power density depends on the flow of fluid through
the fuel cell. Generally, increasing the rate of flow increases the
output power density.

Electrodes

[0026] The anode 102 and cathode 104 can have a variety of forms and be
made from a variety of materials. For example, the anode and/or cathode
can be formed as plates (as shown in FIG. 1), mesh (as shown in FIG. 2),
tubes (as shown in FIG. 3), or other shapes of conductive material. The
anode and/or cathode can also be a conductive film formed over an inert
non-conducting base material formed in the shape of, for example, a
plate, tube, or mesh. The conductive films can be formed on the
non-conducting base material by a variety of methods, including, for
example, sputtering, physical vapor deposition, plasma deposition,
chemical vapor deposition, screen printing, and other coating methods.

[0027] The anode 102 and cathode 104 are formed using a conductive
material, such as, for example, metal, carbon, conductive polymer, or
metallic compound. Suitable conductive materials are typically
non-corroding and can include, for example, gold, vitreous carbon,
graphite, platinum, ruthenium dioxide, and palladium, as well as other
materials known to those skilled in the art. Suitable non-conducting base
materials for use with a conductive film include plastic and polymeric
materials, such as, for example, polyethylene, polypropylene,
polyurethanes, and polyesters. It will be understood that the anode 102
and cathode 104 of any particular embodiment are not necessarily made
using the same materials.

[0028] The conductive material and/or the optional non-conducting base
material are often porous or microporous. For example, the conductive
material and/or the optional non-conducting base material may be formed,
for example, as a mesh, a reticulated structure (e.g., reticulated
graphite), a microporous film, or a film that is permeable to the anode
reductant and/or cathode oxidant. The surface area of the electrode can
also be increased by roughening. Preferably, the actual exposed surface
area of the anode and/or cathode is larger than the macroscopic geometric
surface area because the anode and/or cathode are reticulated, mesh,
roughened, porous, microporous, and/or fibrous. In addition, the
conductive material and/or the optional non-conducting base material can
be an ion selective membrane.

[0029] FIGS. 1 to 5 illustrate a few of the possible configurations for
the anode 102 and cathode 104. The anode 102 and cathode 104 can be
formed as plates and separated by optional non-conducting spacers 103, as
illustrated in FIG. 1. The plates can be, for example, permeable or
non-permeable plates of conductive material that are optionally formed on
a base material. The fuel cell 100 may be configured, for example, so
that biological fluid flows through and/or between the spacers 103 and
then between the anode 102 and the cathode 104, or the fuel cell 100 may
be configured so that biological fluid flows through a permeable anode
102 and permeable cathode 104.

[0030] Another embodiment of a fuel cell 200 includes an anode 202 and a
cathode 204 formed out of a woven or mesh material, as shown in FIG. 2.
The anode 202 and cathode 204 can be separated by a woven or mesh
non-conducting spacer 203.

[0031] Yet another embodiment of a fuel cell 300 includes an anode 302 and
a cathode 304 formed as tubes and separated by an optional non-conducting
spacer 303, as shown in FIG. 3. The anode 302 and cathode 304 of this
fuel cell 300 are formed, for example, using a permeable or mesh material
to allow flow of a biological fluid through the anode 302 and/or cathode
304. As an alternative, the fuel cell 300 may be configured for fluid
flow between or through the spacers 303 and between the anode 302 and
cathode 304. In addition, instead of individual tubes, the anode 302 and
cathode 304 may form spirals.

[0032] Another embodiment of a fuel cell 400 includes a tubular anode 402
with one or more planar cathode plates 404 in the center, as shown in
FIG. 4. The tubular anode 402 and planar cathode plates 404 may be
separated by an optional tubular spacer 403. Again, the fuel cell 400 may
be configured, for example, so that biological fluid flows through a
permeable anode and cathode or so that biological fluid flows between the
anode and/or cathode. One alternative embodiment has a tubular cathode
with one or more intersecting planar anode plates at the center.

[0033] Another embodiment of a fuel cell 500 includes a tubular anode 502
with a planar cathode plate 504 at the center and a wider planar spacer
503 intersecting the cathode plate 504 and positioned within the tubular
anode 502 to keep the cathode plate 504 and tubular anode 502 spaced
apart, as shown in FIG. 5. Again, the fuel cell 500 may be configured,
for example, so that biological fluid flows through a permeable anode
and/or cathode or so that biological fluid flows between the anode and
cathode. One alternative embodiment has a tubular cathode with a planar
anode plate at the center.

Redox Polymers

[0034] Water, which is typically the primary mass transporting medium in
many biological systems, is an electrical insulator. Although the
solubility of many compounds is high in water, these compounds can not be
electrolyzed in the absence of transport of electrons through the aqueous
medium. This can be accomplished using a redox polymer, and particularly
a redox hydrogel. Redox polymers generally provide for adequate transport
of electrons if the redox polymer includes active redox functional groups
that are mobile and can carry electrons between the analyte and the
electrode. For example, a redox hydrogel typically contains a large
amount of water. Water soluble reactants and products often permeate
through the redox hydrogel nearly as fast as they diffuse through water.
Electron conduction in the redox hydrogel is through electron exchange
between polymer segments that are mobile after the polymer is hydrated.

[0035] The anode redox polymer and cathode redox polymer are deposited on
the anode 104 and cathode 102, respectively. In general, the redox
polymers include electroreducible and electrooxidizable ions,
functionalities, species, or molecules having redox potentials.
Preferably, these redox potentials are well-defined. The redox potentials
of the redox hydrogels are typically within a range at which water is
neither electrooxidized or electroreduced. At neutral pH and 25°
C., this range is from about (-)0.65 V to about (+) 0.58 V versus the
standard calomel electrode (SCE) (i.e., from about (-)0.42 V to about
(+)0.81 V versus the standard hydrogen electrode (SHE)). The preferred
range of the redox potential for the anode redox polymer is from about
-0.65 V to about +0.05 V (SCE). The preferred range of the redox
potential for the cathode redox polymer is from about +0.3 V to about
+0.7 V (SCE).

[0036] The preferred redox polymers include a redox species bound to a
polymer which can in turn be immobilized on the working electrode. In
general, redox polymers suitable for use in the invention have structures
or charges that prevent or substantially reduce the diffusional loss of
the redox species during the period of time that the sample is being
analyzed. The bond between the redox species and the polymer may be
covalent, coordinative, or ionic. Examples of useful redox polymers and
methods for producing them are described in U.S. Pat. Nos. 5,262,035;
5,262,305; 5,320,725; 5,264,104; 5,264,105; 5,356,786; 5,593,852; and
5,665,222, incorporated herein by reference. Although any organic or
organometallic redox species can be bound to a polymer and used as a
redox polymer, the preferred redox species is a transition metal compound
or complex. The preferred transition metal compounds or complexes include
osmium, ruthenium, iron, and cobalt compounds or complexes. In the
preferred complexes, the transition metal is coordinatively bound to one
or more ligands and covalently bound to at least one other ligand. The
ligands are often mono-, di-, tri-, or tetradentate. The most preferred
ligands are heterocyclic nitrogen compounds, such as, for example,
pyridine and/or imidazole derivatives. For example, the multidentate
ligands typically include multiple pyridine and/or imidazole rings.
Alternatively, polymer-bound metallocene derivatives, such as, for
example, ferrocene, can be used. An example of this type of redox polymer
is poly(vinylferrocene) or a derivative of poly(vinylferrocene)
functionalized to increase swelling of the redox polymer in water.

[0037] Another type of redox polymer contains an ionically-bound redox
species. Typically, this type of mediator includes a charged polymer
coupled to an oppositely charged redox species. Examples of this type of
redox polymer include a negatively charged polymer such as Nafion®
(DuPont) coupled to multiple positively charged redox species such as an
osmium or ruthenium polypyridyl cations. Another example of an
ionically-bound mediator is a positively charged polymer such as
quaternized poly(4-vinyl pyridine) or poly(1-vinyl imidazole) coupled to
a negatively charged redox species such as ferricyanide or ferrocyanide.
The preferred ionically-bound redox species is a multiply charged, often
polyanionic, redox species bound within an oppositely charged polymer.

[0038] In another embodiment of the invention, suitable redox polymers
include a redox species coordinatively bound to a polymer. For example,
the mediator may be formed by coordination of an osmium, ruthenium, or
cobalt 2,2'-bipyridyl complex to poly(1-vinyl imidazole) or poly(4-vinyl
pyridine) or by co-polymerization of, for example, a
4-vinyl-2,2'-bipyridyl osmium, ruthenium, or cobalt complex with 1-vinyl
imidazole or 4-vinylpyridine.

[0039] Examples of other redox species include stable quinones and species
that in their oxidized state have quinoid structures, such as Nile blue
and indophenol. A preferred tetrasubstituted quinone usually has carbon
atoms in the positions neighboring the oxygen-containing carbon.

[0040] The preferred redox species are osmium or ruthenium transition
metal complexes with one or more ligands, each ligand having one or more
nitrogen-containing heterocycles. Examples of such ligands include
pyridine, imidazole rings, and ligands that include two or more pyridine
and/or imidazole rings such as, for example, 2,2'-bipyridine;
2,2',2''-terpyridine; 1,10-phenanthroline; ligands having the following
structures:

##STR00001##

and derivatives thereof.

[0041] Suitable derivatives of these ligands include, for example, the
addition of alkyl, alkoxy, vinyl, allyl, vinylester, and acetamide
functional groups to any of the available sites on the heterocyclic ring,
including, for example, on the 4-position (i.e., para to nitrogen) of the
pyridine rings or on one of the nitrogen atoms of the imidazole.
Typically, the alkyl, alkoxy, vinyl, and acetamide functional groups are
C1 to C6 and, preferably, C1 to C3 functional groups (referring to the
number of carbon atoms in the functional group). Suitable derivatives of
2,2'-bipyridine for complexation with the osmium cation include, for
example, mono-, di-, and polyalkyl-2,2'-bipyridines, such as
4,4'-dimethyl-2,2'-bipyridine, and mono-, di-, and
polyalkoxy-2,2'-bipyridines, such as 4,4'-dimethoxy-2,2'-bipyridine.
Suitable derivatives of 1,10-phenanthroline for complexation with the
osmium cation include, for example, mono-, di-, and
polyalkyl-1,10-phenanthrolines, such as 4,7-dimethyl-1,10-phenanthroline,
and mono, di-, and polyalkoxy-1,10-phenanthrolines, such as
4,7-dimethoxy-1,10-phenanthroline. Suitable derivatives for
2,2',2''-terpyridine include, for example, mono-, di-, tri-, and
polyalkyl-2,2',2''-terpyridines, such as
4,4',4''-trimethyl-2,2',2''-terpyridine, and mono-, di-, tri-, and
polyalkoxy-2,2',2''-terpyridines, such as
4,4',4''-trimethoxy-2,2',2''-terpyridine.

[0042] Typically, the alkyl and alkoxy groups are C1 to C6 alkyl or
alkoxy, and, preferably, C1 to C3 alkyl or alkoxy.

[0043] Suitable redox species include, for example, osmium cations
complexed with (a) two bidentate ligands, such as 2,2'-bipyridine,
1,10-phenanthroline, or derivatives thereof (the two ligands not
necessarily being the same), (b) one tridentate ligand, such as
2,2',2''-terpyridine and 2,6-di(imidazol-2-yl)-pyridine, or (c) one
bidentate ligand and one tridentate ligand. Suitable osmium transition
metal complexes include, for example, [(bpy)2OsCl].sup.+/2+,
[(dimet)2OsCl].sup.+/2+, [(dmo)2OsCl].sup.+/2+,
[terOsCl2]0/+, [trimetOsCl2]0/+, and
[(ter)(bpy)Os]2+/3+ where bpy is 2,2'-bypyridine, dimet is
4,4'-dimethyl-2,2'-bipyridine, dmo is 4,4'-dimethoxy-2,2'-bipyridine, ter
is 2,2',2''-terpyridine, and trimet is
4,4',4''-trimethyl-2,2',2''-terpyridine.

[0044] The redox species often exchange electrons rapidly between each
other and the electrode so that the complex can be rapidly oxidized
and/or reduced. In general, iron complexes are more oxidizing than
ruthenium complexes, which, in turn, are more oxidizing than osmium
complexes. In addition, the redox potential generally increases with the
number of coordinating heterocyclic rings.

[0045] Typically, the polymers used for the redox polymers have
nitrogen-containing heterocycles, such as pyridine, imidazole, or
derivatives thereof for binding as ligands to the redox species. Suitable
polymers for complexation with redox species, such as the transition
metal complexes, described above, include, for example, polymers and
copolymers of poly(1-vinyl imidazole) (referred to as "PVI") and
poly(4-vinyl pyridine) (referred to as "PVP"), as well as polymers and
copolymer of poly(acrylic acid) or polyacrylamide that have been modified
by the addition of pendant nitrogen-containing heterocycles, such as
pyridine and imidazole. Modification of poly(acrylic acid) may be
performed by reaction of at least a portion of the carboxylic acid
functionalities with an aminoalkylpyridine or aminoalkylimidazole, such
as 4-ethylaminopyridine, to form amides. Suitable copolymer substituents
of PVI, PVP, and poly(acrylic acid) include acrylonitrile, acrylamide,
acrylhydrazide, and substituted or quaternized N-vinyl imidazole. The
copolymers can be random or block copolymers.

[0046] The transition metal complexes typically covalently or
coordinatively bind with the nitrogen-containing heterocycles (e.g.,
imidazole and/or pyridine) of the polymer. Alternatively, the transition
metal complexes may have vinyl functional groups through which the
complexes can be co-polymerized with vinylic heterocycles, amides,
nitriles, carboxylic acids, sulfonic acids, or other polar vinylic
compounds, particularly, for those compounds whose polymer is known to
dissolve or swell in water.

[0047] Typically, the ratio of osmium or ruthenium transition metal
complex to imidazole and/or pyridine groups ranges from 1:10 to 1:1,
preferably from 1:2 to 1:1, and more preferably from 3:4 to 1:1.
Generally, the redox potentials of the hydrogels depend, at least in
part, on the polymer with the order of redox potentials being
poly(acrylic acid)<PVI<PVP.

[0048] A variety of methods may be used to immobilize a redox polymer on
an electrode surface. One method is adsorptive immobilization. This
method is particularly useful for redox polymers with relatively high
molecular weights. The molecular weight of a polymer may be increased,
for example, by cross-linking. The polymer of the redox polymer may
contain functional groups, such as, for example, hydrazide, amine,
alcohol, heterocyclic nitrogen, vinyl, allyl, and carboxylic acid groups,
that can be crosslinked using a crosslinking agent. These functional
groups may be provided on the polymer or one or more of the copolymers.
Alternatively or additionally, the functional groups may be added by a
reaction, such as, for example, quaternization. One example is the
quaternization of PVP with bromoethylamine groups.

[0049] Suitable cross-linking agents include, for example, molecules
having two or more epoxide (e.g., poly(ethylene glycol) diglycidyl ether
(PEGDGE)), aldehyde, aziridine, alkyl halide, and azide functional groups
or combinations thereof. Other examples of cross-linking agents include
compounds that activate carboxylic acid or other acid functional groups
for condensation with amines or other nitrogen compounds. These
cross-linking agents include carbodiimides or compounds with active
N-hydroxysuccinimide or imidate functional groups. Yet other examples of
cross-linking agents are quinones (e.g., tetrachlorobenzoquinone and
tetracyanoquinodimethane) and cyanuric chloride. Other cross-linking
agents may also be used. In some embodiments, an additional cross-linking
agent is not required. Further discussion and examples of cross-linking
and cross-linking agents are found in U.S. Pat. Nos. 5,262,035;
5,262,305; 5,320,725; 5,264,104; 5,264,105; 5,356,786; and 5,593,852,
herein incorporated by reference.

[0050] In another embodiment, the redox polymer is immobilized by the
functionalization of the electrode surface and then the chemical bonding,
often covalently, of the redox polymer to the functional groups on the
electrode surface. One example of this type of immobilization begins with
a poly(4-vinylpyridine). The polymer's pyridine rings are, in part,
complexed with a reducible/oxidizable species, such as
[Os(bpy)2Cl].sup.+/2+ where bpy is 2,2'-bipyridine. Part of the
pyridine rings are quaternized by reaction with 2-bromoethylamine. The
polymer is then crosslinked, for example, using a diepoxide, such as
poly(ethylene glycol) diglycidyl ether.

[0051] Carbon surfaces can be modified for attachment of a redox species
or polymer, for example, by electroreduction of a diazonium salt. As an
illustration, reduction of a diazonium salt formed upon diazotization of
p-aminobenzoic acid modifies a carbon surface with phenylcarboxylic acid
functional groups. These functional groups can be activated by a
carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC). The activated functional groups are bound with a
amine-functionalized redox couple, such as, for example, the quaternized
osmium-containing redox polymer described above or 2-aminoethylferrocene,
to form the redox couple.

[0052] Similarly, gold and other metal surfaces can be functionalized by,
for example, an amine, such as cystamine, or by a carboxylic acid, such
as thioctic acid. A redox couple, such as, for example,
[Os(bpy)2(pyridine-4-carboxylate)Cl]0/+, is activated by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to
form a reactive O-acylisourea which reacts with the gold-bound amine to
form an amide. The carboxylic acid functional group of thioctic acid can
be activated with EDC to bind a polymer or protein amine to form an
amide.

Enzymes

[0053] The enzymes of the anode and cathode electrolysis layers catalyze
an electrochemical reaction of an anode reductant or cathode oxidant,
respectively. Typically, different enzymes are provided in the anode and
cathode electrolysis layers. In some embodiments, more than one enzyme is
provide in the anode and/or cathode electrolysis layers. A variety of
enzymes are useful including, for example, laccase and cytochrome C
oxidase on the cathode for electroreduction of oxygen; peroxidases on the
cathode for electroreduction of hydrogen peroxide; oxidases and
dehydrogenases on the anode for electrooxidation of glucose, lactate, and
other biochemicals; pyranose oxidase on the anode for electrooxidation of
D-glucose, L-sorbose, and D-xylose; and glucose oxidase, oligosaccharide
dehydrogenase, or pyrroloquinoline quinone (PQQ) glucose dehydrogenase on
the anode for electrooxidation of glucose. These enzymes, preferably, do
not include leachable co-factors, such as NAD+ and NADP+. Other enzymes
may also be included, particularly on or near the anode, to convert more
complex molecules, such as starches and cellulose, into sugars, alcohols,
and/or carboxylic acids.

[0054] One category of suitable enzymes includes thermostable enzymes
which are defined herein, unless otherwise indicated, as enzymes that
function for one day, and, preferably, for five days, or more at
37° C., losing 10% or less, and, preferably, 5% or less, of their
activity over the period of use. Examples of thermostable enzyme include
laccase from the thermophilic fungus myceliophthora thermophilic,
cytochrome C perioxidases from thermophilic bacterium PS3 and thermus
thermophilus, peroxidase from soybean, and pyranose oxidase from the
white rot fungus phlebiopsis gigantea. Other commercially available
thermostable enzymes include L-lactate dehydrogenase from bacillus,
malate dehydrogenase from thermus species (expressed in E. coli), glucose
oxidase from aspergillus, microbial pyruvate oxidase, and urate oxidase
from bacillus. Thermostable enzymes that hydrolyze larger biological
molecules into electrooxidizable sugars include, for example,
α-amylase from bacillus stearothermophilus, β-amylase from
aspergillus, glucan-1,4-α-glucosidase from rhizopus niveus,
cellulase from aspergillus niger, endo-1-3(4)-β-glucanase from
aspergillus niger, dextranase from leuconostoc mesenteroides,
α-glucosidase from bacillus stearothermophilus, β-glucosidase
from caldocellum saccharolyticum, β-galactosidase from aspergillus,
β-fructofuranosilidase from yeast, and lactase from aspergillus
oryzae.

[0055] Alternatively, the enzyme is immobilized in a non-conducting
inorganic or organic polymeric matrix to increase the thermostablity of
the enzyme. Discussion regarding immobilization of an enzyme in an
inorganic polymeric matrix is found in U.S. patent application Ser. No.
08/798,596, now issued as U.S. Pat. No. 5,972,199, and PCT Patent
Application No. US98/02403, now published as PCT Publication WO 98/35053,
incorporated herein by reference. A sol-gel polymerization process
provides a method for the preparation of an inorganic polymeric matrix
(e.g., glass) by the polymerization of suitable monomers at or near
room-temperature. Suitable monomers include, for example, alkoxides and
esters of metallic and semiconducting elements, with preferred metallic
and semiconducting elements including Si, Al, Ti, Zr, and P. The most
preferred monomers include silicon and have a silicon to oxygen ratio
from about 1:2 to about 1:4.

[0056] For example, enzymes can be immobilized in silica polymeric
matrices made by sol-gel processes, such as the hydrolysis of
tetramethoxysilane or another polyalkoxysilane that contains one or more
silicon atoms. Condensation of the resulting silanol in the presence of
the enzyme results in entrapment of the enzyme. This process has been
referred to as sol-gel immobilization. Binding of enzymes in silica or
other inorganic polymeric matrices formed from sol-gels can stabilize the
enzyme. Entrapment of glucose oxidase, a glycoprotein, in a silica
sol-gel matrix greatly improves the stability of the enzyme, which
retains activity when heated in water to 98° C. for 10 minutes.

[0057] An enzyme stabilized by the silica sol gel matrix can be ground to
a fine powder and dispersed in a silicone, preferably in an elastomeric
silicone, and most preferably in a water-based elastomeric silicone
precursor. This dispersion is then applied to the cathode as a binder of
the enzyme. The binder preferably includes material to extract and store
oxygen from the environment. Silicone is a preferred binder in this layer
due to its ability to dissolve oxygen and its oxygen permeability.
Elastomeric silicones are preferred because of high oxygen solubility.

[0058] The stability of an enzyme in an inorganic polymeric matrix
depends, at least in part, on the ionic characteristics of the enzyme and
those of the immobilizing, often inorganic, polymeric matrix. A hydrated
silica gel has an isoelectric point (pI) (i.e., the pH at which the net
charge on the molecule is zero) near pH 5. Glucose oxidase, with pI=3.8,
retained its activity upon sol-gel immobilization and was stabilized when
immobilized in the hydrated silica gel matrix so that the half-life of
the enzyme was increased by about 200-fold at 63° C. Lactate
oxidase (pI=4.6) and glycolate oxidase (pI≈9.6), on the other
hand, each lost at least 70% of their activity upon immobilization in a
hydrated silica gel and the stability of these two enzymes was not
greatly improved.

[0059] In contrast to the loss of activity of these enzymes in hydrated
silica alone, when poly(1-vinyl imidazole) (PVI) (a weak base) or
poly(ethyleneimine) (PEI) (a stronger base) was used to form an adduct in
the hydrated silica gel, the half-life of lactate oxidase (pI=4.6)
increased more than 100-fold at 63° C. and the enzyme was
immobilized without significant loss of activity. The adduct can be
formed by, for example dissolving lactate oxidase in an aqueous buffer
solution in which poly(1-vinyl imidazole) is co-dissolved, and the
lactate oxidase-poly(1-vinyl imidazole) mixture is immobilized in silica
by the sol-gel process, a stable, immobilized lactate oxidase is
obtained. The stabilized lactate oxidase can be heated in water to
90° C. for 10 minutes and still retain enzymatic activity. A
similar adduct which retains enzymatic activity can be formed with
poly(ethyleneimine).

[0060] Formation of an adduct between glycolate oxidase (pI≈9.6)
and poly(1-vinyl imidazole) (a weak base) did not improve the stability
of the enzyme, but the formation of an adduct between glycolate oxidase
(pI≈9.6), and poly(ethylene imine) (a stronger base) increased
the half-life of glycolate oxidase more than 100-fold at 60° C.
Forming an adduct between glucose oxidase pI=3.8 and PVI or PEI did not
further improve the stability of the enzyme after its immobilization in
hydrated silica.

[0061] It is thought that these functionally essential, positively charged
surface residues (e.g., arginine) of the lactate and glycolate oxidases
may interact with negatively charged polysilicate anions of the hydrated
silica, resulting in a decrease in activity upon sol-gel immobilization.
However, when the enzyme surface is enveloped by a flexible polycation
buffer (i.e., PVI and/or PEI, depending on the isoelectric point of the
enzyme) then the polysilicate anions interact with the cationic buffer
molecules, and not with the cationic residues of the enzyme, thereby
stabilizing the enzyme by encasement in the silica gel. Thus, it is
thought that PVI and PEI form adducts, acting as polycationic buffers for
enzymes such as lactate oxidase. PEI also acts as a cationic buffer for
enzymes such as glycolate oxidase. It is thought that PVI is not an
effective buffer for glycolate oxidase, because glycolate oxidase is a
stronger base.

[0062] In general, the addition of a polycation, such as, for example,
poly(1-vinyl imidazole) or poly(ethyleneimine), prior to sol-gel
immobilization stabilizes the enzyme. Preferably, the added polycation is
a more basic polyelectrolyte than the enzyme. Enzymes with high
isoelectric points often need more basic polyelectrolytes for
stabilization. Poly(ethyleneimine) is more basic than poly(1-vinyl
imidazole).

[0063] Poly(1-vinyl imidazole), a polycation at pH 7, binds at this pH to
enzymes such as lactate oxidase, that are polyanions at pH 7. Thus, the
addition of a particular polymer to a particular enzyme can greatly
increase the stability the enzyme. In the case of lactate oxidase,
addition of poly(ethyleneimine), also a polybasic polymer and also
multiply protonated at pH 7, in place of poly(1-vinyl imidazole) improved
stability of the enzyme, although not as much as the addition of the
preferred polymer, poly(1-vinyl imidazole). The stabilized enzyme can
then be used at higher temperatures and/or for longer durations than
would be possible if the enzyme were immobilized alone in the sol-gel.

[0064] The sol gel matrix in which an enzyme is immobilized and stabilized
is often not an electron conductor. The matrix can be modified by
binding, often through covalent bonds, a redox functional group to the
matrix or its precursor. Examples of suitable redox functional groups
include the redox species described above for use in the redox polymer,
including, for example, osmium, ruthenium, and cobalt complexes having
ligands including one or more pyridine and/or imidazole rings. Moreover,
the redox functional group preferably includes a spacer arm covalently or
coordinatively attached a metal cation of the redox functional group or
one of the ligands. One end of the spacer arm is covalently linked to,
for example, silicon atoms of the matrix. The other end of the spacer arm
is covalently or coordinatively linked to the redox functional group. The
enzyme can be immobilized in such a matrix and electrons can be exchanged
between the enzyme and the electrode using the redox functional group
coupled to the matrix.

[0065] In some embodiments, non-corroding, electron-conducting particles
are disposed within the matrix to increase the conductivity of the
matrix; particularly, for those matrices that include attached redox
functional groups. Examples of such particles include graphite, carbon
black, gold, and ruthenium dioxide. Typically, these particles have a
diameter of1 μm or less and a surface area of 1 m2/g or more,
preferably, 10 m2/g or more, and, more preferably, 100 m2/g or
more. Alternatively, VOCl3 can be hydrolyzed to form a polymeric
matrix, that, when reduced, is conducting.

[0066] In other embodiments, an enzyme is immobilized and stabilized in a
sol gel matrix and the enzyme catalyzes a reaction of a chemical to form
a product that is subsequently electrooxidized or electroreduced in the
presence of a second enzyme that is electrically coupled to an electrode.
For example, glucose can react in the presence of glucose oxidase that is
stabilized in a sol gel matrix to form gluconolactone and hydrogen
peroxide. The hydrogen peroxide diffuses out of the sol gel matrix to the
proximity of the cathode and is electroreduced to water by a thermostable
enzyme, such as soybean peroxidase.

[0068] One embodiment of the anode is formed using a high surface area
graphite fiber/carbon black electrode using polypropylene or
polytetrafluoroethylene (e.g., Teflon®) as a binder. The anode redox
polymer and anode enzyme are then disposed on the anode.

[0069] The anode potential can be limited by the (a) redox potential of
the anode enzyme, (b) the concentration of the anode reductant at the
anode, and (c) the redox potential of the anode redox polymer. Reported
redox potentials for known anode enzymes range from about -0.4 V to about
-0.5 V versus the standard calomel electrode (SCE). Typically, the
preferred anode redox polymers have a redox potential that is at least
about 0.1 V positive of the redox potential of the anode enzyme. Thus,
the preferred anode redox polymer can have a redox potential of, for
example, about -0.3 V to -0.4 V (SCE), however, the potential of the
anode redox polymer may be higher or lower depending, at least in part,
on the redox potential of the anode redox enzyme. Preferred anode redox
polymers for the anode include [(dmo)2OsCl].sup.+/2,
[terOsCl2]0/+, and [trimetOsCl2]0/+ coupled to either
PVI or poly(acrylic acid) or a copolymer of 4-vinyl pyridine or 1-vinyl
imidazole.

[0070] In some embodiments, one or more additional enzymes are provided in
proximity to or disposed on the anode. The additional enzyme or enzymes
break down starch, cellulose, poly- and oligosaccharides, disaccharides,
and trisaccharides into the sugars, alcohols, and/or carboxylic acids
that are used as fuel. Examples of such catalysts include α-amylase
from bacillus stearothermophilus, β-amylase from aspergillus,
glucan-1,4-α-glucosidase from rhizopus niveus, cellulase from
aspergillus niger, endo-1-3(4)-β-glucanase from aspergillus niger,
dextranase from leuconostoc mesenteroides, α-glucosidase from
bacillus stearothermophilus, β-glucosidase from caldocellum
saccharolyticum, β-galactosidase from aspergillus,
β-fructofuranosilidase from yeast, and lactase from aspergillus
oryzae.

Cathode

[0071] In one embodiment of the fuel cell, the cathode reduces gaseous
O2 that is typically dissolved in the biological fluid or
originating from the air. In another embodiment of the fuel cell,
hydrogen peroxide is formed in a non-enzyme-catalyzed electrode reaction
or in an enzyme-catalyzed reaction on or off the cathode and then the
hydrogen peroxide is electroreduced at the cathode. Preferred cathode
enzymes for the reduction of O2 and H2O2 include, for
example, tyrosinase, horseradish peroxidase, soybean peroxidase, other
peroxidases, laccases, and/or cytochrome C peroxidases.

[0072] One embodiment of the cathode includes a porous membrane formed
over at least a portion of cathode. The porous membrane has an O2 or
H2O2 permeable, hydrophobic outer surface and an O2 or
H2O2 permeable hydrophilic inner surface. In another
embodiment, the cathode includes an outer layer of a hydrophobically
modified porous silicate carbon composite, formed of an
alkyltrialkoxysilane precursor, and carbon black. The inner layer is a
hydrophilic silca-carbon composite. In another embodiment, the electrode
is a microporous Teflon PTFE bound acetylene/carbon black electrode. The
inner surface is plasma processed to make it hydrophilic. The redox
polymer and enzyme are deposited on the inner surface of the cathode.
When the cathode is exposed to O2 originating in blood or a body
fluid, the cathode may only include hydrophilic surfaces in contact with
the O2 transporting biological fluid.

[0073] The cathode potential can be limited by the (a) redox potential of
the cathode enzyme, (b) the concentration of the cathode oxidant at the
cathode, and (c) the redox potential of the cathode redox polymer.
Reported redox potentials for known O2 reducing enzymes range from
about +0.3 V to about +0.6 V versus the standard calomel electrode (SCE).
Typically, the preferred cathode redox polymer has a redox potential that
is at least about 0.1 V negative of the redox potential of the enzyme.
Thus, the preferred redox polymer has redox potential of, for example,
about +0.4 to +0.5 V (SCE), however, the potential of the cathode redox
polymer may be higher or lower depending, at least in part, on the redox
potential of the cathode redox enzyme.

[0074] For osmium complexes used as the cathode redox polymer, typically,
at least four, usually, at least five, and, often, all six of the
possible coordination sites of the central osmium atom are occupied by
nitrogen atoms. Alternatively, for complexes of ruthenium used as the
cathode redox polymer, typically, four or fewer, and, usually, three or
fewer of the possible coordination sites are nitrogen occupied. Preferred
cathode redox polymers include [(ter)(bpy)Os]2+/3+ coupled to PVI or
PVP.

Non-Fouling Coatings

[0075] An optional non-fouling coating is formed over at least that
portion of the electrodes of the fuel cell. The non-fouling coating
prevents or retards the penetration of macromolecules, such as proteins,
having a molecular weight of 5000 daltons or more, into the electrodes of
the fuel cell. This can be accomplished using a polymeric film or coating
having a pore size that is smaller than the biomolecules that are to be
excluded or having anionic and/or cationic functional groups that repel
cationic or anionic macromolecules, respectively. Such biomolecules may
foul the electrodes and/or the electrolysis layer thereby reducing the
effectiveness of the fuel cell and altering the expected electrical power
generation. The fouling of the electrodes may also decrease the effective
life of the fuel cell.

[0076] For example, the electrodes of the fuel cell may be completely or
partially coated on their exterior with a non-fouling coating. A
preferred non-fouling coating is a polymer, such as a hydrogel, that
contains at least 20 wt. % fluid when in equilibrium with the
analyte-containing fluid. Examples of suitable polymers are described in
U.S. Pat. No. 5,593,852, incorporated herein by reference, and include
crosslinked polyethylene oxides, such as polyethylene oxide tetraacrylate
and diacrylate. For example, polyethylene oxide ("PEO") chains, typically
of 8-18 kilodaltons are terminally modified with reactive groups, such as
acrylates and methacrylates. In addition, diesters of PEO can be reacted
with star-dendrimer PEO polyamines to form the non-fouling coatings.

Implantation in a Blood Vessel

[0077] For continuously producing power, the reactant-carrying fluid
typically flows through the fuel cell, so as to replenish the anode
reductant and/or cathode oxidant exhausted by reacting at the anode
and/or cathode, respectively. When the fuel cell is implanted in a blood
vessel of an animal (e.g., human, mammal, bird, or fish), the fluid may
be pumped through the fuel cell by the heart, obviating any need for a
mechanical pump and thereby reducing the weight and volume of the system
containing the fuel cell.

Use and Storage of Electrical Power Generated by the Fuel Cell

[0078] The electrical power generated by the fuel cell can be used to
operate a variety of devices, including, for example, medical or other
devices implanted in a human or animal. Examples of medical devices
include pacemakers, nerve growth stimulators, nerve stimulators for
relief of chronic pain, stimulators for regrowth of bone or other tissue,
drug-release valves or microvalves, and fluid-flow control valves, such
as a valve in a duct or in the urinary tract. The electrical power can
also be used to operate external devices connected to the fuel cell
(e.g., a fuel cell implanted in a plant or tree). In addition, the
electrical power can be stored in a storage device, such as a capacitive
storage element or battery, for later use.

[0079] The present invention should not be considered limited to the
particular examples described above, but rather should be understood to
cover all aspects of the invention as fairly set out in the attached
claims. Various modifications, equivalent processes, as well as numerous
structures to which the present invention may be applicable will be
readily apparent to those of skill in the art to which the present
invention is directed upon review of the instant specification.